Anti-Inflammatory Botanical Extract

ABSTRACT

A botanical extract that exhibits anti-inflammatory activity, wherein the botanical extract is at least an extract from the genus Anacardium.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of pending U.S.patent application Ser. No. 16/515,104, filed 18 Jul. 2019 (nowallowed), which claims the benefit of U.S. Patent Application No.62/725,461, filed 31 Aug. 2018, the disclosure of which is incorporatedherein in its entirety by reference.

BACKGROUND OF THE INVENTION

Field of the Invention. The present invention generally relates to abotanical extract that exhibits anti-inflammatory activity, andcompositions comprising such an extract.

Arachidonic acid and its metabolites are important mediators ofinflammation. Arachidonic acid (‘AA’) is a component of membranephospholipids where the rate-limiting step in the formation of itsmetabolites depends on its release from the cell membrane phospholipidpool mediated through activation of phospholipases. Thereafter, it canbe metabolized by one of two pathways—by cyclooxygenase (‘COX’) to yieldeicosanoids such as prostaglandins (‘PGE2’), prostacyclins, andthromboxanes, or it can be metabolized by 5-lipoxygenase (‘5-LOX’) toresult in the production of leukotrienes and lipoxins. These eicosanoidsserve as intracellular messengers and play significant roles in theregulation of signal transduction in pain and inflammatory responses. Anillustration of the arachidonic acid metabolism pathway is provided inFIG. 1.

Cyclooxygenase—a prostanoid synthase also known asprostaglandin-endoperoxide synthase (PTGS, EC 1.14.99.1)—is an enzymethat is responsible for the formation of important biological mediatorscalled prostanoids, including prostaglandins, prostacyclin andthromboxane. COX is the central enzyme in the biosynthetic pathway toprostanoids from arachidonic acid. There are two known isoenzymes—COX-1and COX-2. COX-1 represents the constitutive isoform responsible forproduction of prostaglandins involved in physiological functions such asprotection of the gastric mucosa and maintenance of renal perfusion.COX-2 is not expressed under normal conditions in most cells, butelevated levels are found during inflammation. COX-2 is the dominantisozyme in inflamed tissues, where its induction can be facilitated byseveral pro-inflammatory cytokines, including interleukin-1 (‘IL-1’) andtumor necrosis factor (‘TNF-α’). Pharmacological inhibition of COX bynon-steroidal anti-inflammatory drugs (NSAID) can provide relief fromthe symptoms of inflammation and pain.

Therefore, to prevent the unwanted side effects, it seems practical toinhibit COX-2 selectively for its analgesic and anti-inflammatoryeffects without affecting important physiological processes controlledby the prostaglandins formed by COX-1. Still, there are reports thatassociate the synergistic effect of COX-2 as a constitutive isoenzyme inmaintaining renal blood flow and the glomerular filtration ratesuggesting its selective inhibition may lead to some adverse effects.These effects were experienced by subjects in clinical trials whereinselective COX-2 inhibitors (e.g., celecoxib and rofecoxib) providedsimilar efficacy to that of traditional NSAIDs in osteoarthritis andrheumatoid arthritis pain with better gastric tolerability andequivalent to NSAIDs in renal side effects. Therefore, it is reasonableto assume and have a compound strong enough to cause inhibition of theseisoenzymes yet moderate enough to avoid the unnecessary adverseconsequences, as opposed to a complete selective inhibition of either ofthe enzymes.

Increased expression of COX-2, and hence synthesis of its product PGE2,has also been found to be strongly associated with the induction ofMMP-9, which is a key player in cancer, cardiovascular disease, andinflammation. Therefore, inhibition of COX-2 enzyme may result inregulation of MMP-9 expression and activity that may modulate invasionand migration of cancer cells, prevent or delay the progression ofatherosclerosis and stabilize plaques, regulate macrophage proteinaseexpression, prevent chronic periodontitis and gingivitis, and controlremodeling of liver disease, among others.

The other segment of the Arachidonic acid (‘AA’) metabolism pathway isthrough the 5-lipoxygenase (‘5-LOX’) pathway, where leukotrienes (LTB4,LTC4, LTD4, and LTE4) derived from LTA4 are the end bioactivemetabolites. LTC4 and its products LTD4 and LTE4 act on smooth musclecells of bronchi and blood vessels, where their biologic effects suggesttheir role in allergic reaction and inflammatory processes. For example,in asthma they cause bronchoconstriction, vasoconstriction, andincreased vascular permeability; thus, they are previously known asslow-reacting substances of anaphylaxis. The other component of thispathway—LTB4—is a potent chemotactic factor of neutrophils. While thespecific inhibitor of the 5-LOX enzyme—Zileuton—provides effectiveintervention of asthma attacks where the anti-inflammatory andantibronchospastic effects work together, single therapeutic modalityfor 5-LOX modulators seem insufficient.

Preferably, anti-inflammatory products encompass inhibition of both mainmetabolic pathways of Arachidonic acid (‘AA’) metabolism, possessing awide range of anti-inflammatory activities while also having a bettersafety profile.

Another mediator of inflammation which acts as cytokine and is secretedby immune cells are High Mobility Group Box 1 proteins (‘HMGB1’), alsoknown as high-mobility group protein 1 (‘HMG-1’) and amphoterin. HMGB1is a protein that in humans is encoded by the HMGB1 gene. Like thehistones, HMGB1 is among the most important chromatin proteins. HMGB1 isa 30 kDa nuclear and cytosolic protein, and is a self-derived immuneactivator that has multiple functions in the regulation of immunity andinflammation.

HMGB1 can be released actively by innate immune cells such asmacrophages, monocytes, and dendritic cells at the time of inflammationand injury. For example, macrophages and monocytes actively releaseHMGB1 in a time- and dose-dependent manner in response to stimulationwith exogenous bacterial endotoxin (e.g., lipopolysaccharide, or LPS),or endogenous pro-inflammatory cytokines such as tumor necrosis factor(‘TNF-α’), Interleukin-1 beta (‘IL-1β’), and Interferon gamma (‘IFN-γ’).

HMGB1 can also be released passively by necrotic or damaged cells, andis capable of inducing an inflammatory response by communicating theinsult to the neighboring immune cells, allowing the innate immune cellsto both respond to injury and to further induce inflammation. HMGB1proteins trigger intracellular signaling through receptor for advancedglycosylation end products (‘RAGE’) and/or Toll-like receptors(TLR-2/4), which in turn activate various signaling pathways asmitogen-activated protein kinase (‘MARK’) pathways and subsequentnuclear factor kappa-light-chain-enhancer of activated B cells (‘NF-κB’)mediating inflammation, leading to the expression of various leukocyteadhesion molecules, pro-inflammatory cytokines, and chemokines.

HMGB1 plays significant roles in inflammatory activity and is involvedin a wide range of immune responses. HMGB1 induces maturation andmigration of dendritic cells (‘DCs’), as well as the activation of thesecells and monocytes to produce pro-inflammatory cytokines such as TNF-α,IL-1β, IL-6, and macrophage inflammatory protein 1 (‘MIP-1’). HMGB1 alsoserves as a chemotactic factor for monocytes, macrophages, neutrophils,and DCs to sustain inflammation and elicit innate immune response.

HMGB1 is considered a lead example of a danger signal that originatesfrom the damaged self instead of from invading pathogens. HMGB1 mediatesactivation of innate receptors resulting in the amplification ofinflammatory responses through the release of cytokines, which in turninduce the release of additional HMGB1, further promoting the inductionof these mediators. While pro-inflammatory cytokines such as TNF-α,IL-1β, and IFN-γ are known to mediate the early phases of inflammation,HMGB1 is considered as the late phase dictator in sepsis and tissueinjury.

Targeting HMGB1 may be a pragmatic approach for therapeuticinterventions in inflammatory diseases as it has been identified as acrucial mediator in the pathogenesis of many diseases, including sepsis,arthritis, cancer, and diabetes. For example, the level of HMGB1 hasbeen found to be elevated in (1) synovial fluid of patients withrheumatoid arthritis, (2) septic patients who did not survive comparedto those who did survive, (3) invasion and metastasis of solid tumors,and (4) diabetes and its complications.

As a consequence, many pharmacologic agents have been studied for theirpotential to inhibit release of HMGB1 or HMGB1 activity (see, FIG. 2).These include traditional herbal medicines such as aqueous extracts ofdong guai or dang gui (“female ginseng”—Angelica sinensis), Green tea(Camellia sisensis), and Danshen (“red sage” or “Chinese sage”—Salivamiltorrhiza), which have been found to inhibit endotoxin-induced HMGB1release, as well as protect animals against experimental sepsis.

Accordingly, phytomedicine plays an important role in the management ofmost of these diseases, with plants being a potential source of naturalantioxidants. Studies have shown that the consumption of polyphenoliccompounds found in tea, herbs, fruits, and vegetables is associated withlow risk of these diseases. Consequently, there is a growing researchinterest in plants that exhibit anti-inflammatory activity andhealth-promoting phytoconstituents as potential therapeutic agents.Medicinal plants can provide a safe, cost-effective, ecologicalalternative to chemical antioxidants, which can be toxic on prolongedexposure.

The cashew tree (Anacardium occidentale Linn) is originally from theAmazon, and has subsequently been transplanted to India, Eastern Africa,and other countries for cultivation. The tree produces a very peculiarapple or fruit in the form of a swollen peduncle. Externally at the endof this peduncle the cashew nut grows in its own grey coloredkidney-shaped hard shell. This shell has a soft leathery outer skin anda thin hard inner skin referred to as the husk or testa, which surroundsthe kernel. Between these two skins is a honeycomb structure containingthe cashew nut shell liquid. This liquid comprises anacardic acid,cardanol, and cardol, among other ingredients. Anacardic acid is asalicylic acid, while cardanol and cardol are substituted phenols.

The various parts of the fruit have been studied for their uses. Inaddition to being an edible food, the juice from the cashew apple isused in beverages, while the fruit extract has shown benefit in weightmanagement. Cashew nut shell liquid has been extracted for variousindustrial and agricultural applications, include friction linings,paints, laminating resins, rubber compounding resins, cashew cements,polyurethane based polymers, surfactants, epoxy resins, foundrychemicals, chemical intermediates, insecticides, and fungicides. Cashewtesta has been used in tanning materials.

As noted above, there is a need for effective, nontoxic, naturalcompounds with anti-inflammatory activity. The present inventionprovides one such solution.

BRIEF SUMMARY OF THE INVENTION

Provided herein is a botanical extract composition comprising catechins,wherein the extract has been standardized to a catechin content of about15.0 wt % or greater, based on total weight of the extract. Thebotanical extract composition exhibits anti-inflammatory activity andcomprises at least an extract from the genus Anacardium. Preferably thebotanical extract is at least an extract from Anacardium occidentale L.More preferably, the botanical extract is from at least the testa of thefruit of Anacardium occidentale L.

In one embodiment, the present invention is directed towards an extractof the testa of the fruit of Anacardium occidentale L. comprising about15.0 wt % or greater catechins, based on total weight of the extract.

In a further embodiment, the present invention provides a compositioncomprising the botanical extract of the testa of Anacardium occidentaleL, wherein the botanical extract exhibits anti-inflammatory activity.The botanical extract can be present in the composition in an amount ofabout 4 μg/mL or greater. Preferably, the botanical extract is presentin the composition in an amount of about 4 μg/mL to about 2000 μg/mL.More preferably, the botanical extract is present in the composition inan amount of about 15 μg/mL to about 250 μg/mL.

Regarding the anti-inflammatory activity of the botanical extract of thecomposition, in one aspect compositions containing the botanical extractof the testa of Anacardium occidentale L. inhibit COX-1 activity.Preferably for such compositions inhibiting COX-1 activity, thebotanical extract is present in the composition in an amount of about 15μg/mL to about 250 μg/mL.

In another aspect, compositions containing the botanical extract of thetesta of Anacardium occidentale L. inhibit COX-2 activity. Preferablyfor such compositions inhibiting COX-2 activity, the botanical extractis present in the composition in an amount of about 30 μg/mL to about250 μg/mL.

In even another aspect, compositions containing the botanical extract ofthe testa of Anacardium occidentale L. inhibit 5-LOX activity.Preferably for such compositions inhibiting 5-LOX activity, thebotanical extract is present in the composition in an amount of about 32μg/mL to about 250 μg/mL.

In a further aspect, compositions containing the botanical extract ofthe testa of Anacardium occidentale L. inhibit HMGB 1 activity. In oneembodiment for such compositions inhibiting HMGB 1 activity, thebotanical extract is present in the composition in an amount of about 50μg/mL.

In another aspect, compositions containing the botanical extract of thetesta of Anacardium occidentale L. inhibit COX-1 activity, COX-2activity, 5-LOX activity, and HMGB1 activity.

Compositions containing the botanical extract of the testa of Anacardiumoccidentale L. can further comprise a pharmaceutically acceptablecarrier. Non-limiting examples of such compositions include dietarysupplements and topical compositions.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a general illustration of the Arachiconic acid metabolismpathway.

FIG. 2 is a general illustration of HMGB1-mediated pro-inflammatoryresponses at various sites.

FIG. 3 is an HPLC chromatogram of cashew testa extract at 275 nmwavelength over a retention time of from 0 minutes (start) to 20minutes.

FIG. 4 is LC/MS and LC/PDA (wavelengths of 280 and 350 nm) chromatogramsof cashew testa extract.

FIG. 5 is a graph illustrating percentage COX-1 inhibition using cashewtesta extract at various concentrations.

FIG. 6 is a graph illustrating percentage COX-2 inhibition using cashewtesta extract at various concentrations.

FIG. 7 is a graph illustrating percentage 5-LOX inhibition using cashewtesta extract at various concentrations.

FIG. 8 is a bar graph illustrating the detection of HMGB1 (% release) inmacrophage cell culture supernatant at room atmosphere (21% O₂) (‘RA’),95% O₂ (‘O2’) without cashew testa extract, DMSO (‘Vehicle’), positivecontrol sodium salicylate (‘SS 2 μM’), and 95% O₂ with cashew testaextract (‘CT’).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising discovery that thetesta of the cashew (Anacardium occidentale Linn) is substantially highin certain flavonoids. In particularly, it has been discovered that theextract of cashew testa comprises catechin and epicatechin as majorcomponents, as well as procyanidins. Data noted herein demonstrates thatcashew testa extract may have anti-inflammatory applications.

For the present application, the term “composition” refers to a productthat treats, improves, promotes, increases, manages, controls,maintains, optimizes, modifies, reduces, inhibits, or prevents aparticular condition associated with a natural state, biological processor disease or disorder. For example, a composition improves theinhibition of oxidation and/or reduces inflammation, and the like in asubject. The term composition includes, but is not limited to,pharmaceutical (i.e., drug), over-the counter (OTC), cosmetic, food,food ingredient or dietary supplement compositions that include aneffective amount of an extract, at least one component thereof, or amixture thereof. Exemplary compositions include cream, cosmetic lotion,pack or powder, or as an emulsion, lotion, liniment foam, tablets,plasters, granules, or ointment. Compositions can also includebeverages, for example, beverages infused with an effective amount of anextract, or a tea satchel containing an effective amount of an extract.Non-limiting examples of food compositions containing an effectiveamount of an extract include baked goods, protein powders, meatproducts, dairy products, and confectionary.

As used herein, the term “extract” or “botanical extract” refers to asolid, viscid, or liquid substance or preparation that includes one ormore active ingredients of a substance of at least the plant genusAnacardium (e.g., Anacardium humile, Anacardium othonianum, Anacardiumgiganteum, Anacardium nanum, Anacardium negrense, and/or Anacardiumoccidentale), preferably Anacardium occidentale L. Preferably, theactive ingredient is derived from the extract of the testa of thecashew. The extract is prepared using a solvent such as water, loweralcohols of 1 to 4 carbon atoms (e.g., methanol, ethanol, butanol,etc.), ethylene, acetone, hexane, ether, chloroform, ethylacetate,butylacetate, dichloromethane, N,N-dimethylformamide (‘DMF’),dimethylsulfoxide (‘DMSO’), 1,3-butylene glycol, propylene glycol, andcombinations thereof, but also a fraction of the crude extract in such asolvent. So long as it assures the extraction and preservation of theactive ingredient(s), any extraction method may be employed.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” of a pure compound, composition, extract, extractmixture, component of the extract, and/or active agent or ingredient, ora combination thereof refers to an amount effective at dosages and forperiods of time sufficient to achieve a desired result. For example, the“effective amount” or “therapeutically effective amount” refers to thatamount of a pure compound, composition, extract, botanical extract,extract mixture, botanical extract mixture, component of the extract,and/or active agent or ingredient, or a combination thereof of thisinvention which, when administered to a subject (e.g., mammal, such as ahuman), is sufficient to effect treatment, such as improving theinhibition of oxidation and/or reducing inflammation, and the like in asubject. The amount of a composition, extract, botanical extract,extract mixture, botanical extract mixture, component of the extract,and/or active agent or ingredient of this disclosure that constitutes an“effective amount” or “therapeutically effective treatment” will varydepending on the active agent or the compound, the condition beingtreated and its severity, the manner of administration, the duration oftreatment, or the age of the subject to be treated, but can bedetermined routinely by one of ordinary skill in the art having regardto his own knowledge and to this disclosure.

The term “pharmaceutically acceptable” means those drugs, medicaments,extracts or inert ingredients, which are suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,incompatibility, instability, irritation, and the like, commensuratewith a reasonable benefit/risk ratio.

The terms “administer”, “administered”, “administers”, and“administering” are defined as providing a composition to a subject viaa route known in the art, including but not limited to intravenous,intra-arterial, oral, parenteral, buccal, topical, transdermal, rectal,intramuscular, subcutaneous, intraosseous, transmucosal, orintraperitoneal routes of administration. In preferred embodiments, oralroutes of administering a composition are suitable.

As used herein, the term “subject” or “individual” includes mammals towhich a composition may be administered. Non-limiting examples ofmammals include humans, non-human primates, canines, felines, equines,bovines, rodents (including transgenic and non-transgenic mice) or thelike. In some embodiments, the subject is a non-human mammal, and insome embodiments, the subject is human.

As used herein, the term “carrier” refers to a composition that aids inmaintaining one or more plant extracts in a soluble and homogeneousstate in a form suitable for administration, which is nontoxic and whichdoes not interact with other components in a deleterious manner.

Unless indicated otherwise, all proportions and percentages recitedthroughout this disclosure are by weight.

The present invention provides a botanical extract that exhibitsanti-inflammatory activity. More particularly, the present invention isdirected towards a botanical extract of the cashew testa from the genusAnacarium. Such botanical extracts have been found to exhibitanti-inflammatory activity.

As previously stated, useful anti-inflammatory botanical extractsaccording to the present invention include botanical extracts from thegenus Anacardium. More particularly, the extract is a botanical extractchosen from one or more of the species Anacardium humile, Anacardiumothonianum, Anacardium giganteum, Anacardium nanum, Anacardium negrense,and/or Anacardium occidentale. Preferably, the botanical extract is fromthe species Anacardium occidentale L. In one embodiment, the botanicalextract is from the testa of the species Anacardium occidentale L.

Anti-inflammatory compositions according to the present invention mayinclude one or more compounds that may function as active ingredients.The compound may be a component of the botanical extract. For example,the compound can be a phytochemical present in the plant from which theplant extract is obtained. The compound may be at least partiallyresponsible for exhibiting anti-inflammatory activity. The compound canbe any compound capable of inhibiting inflammation. In one embodiment,the compound is chosen from the phytochemicals catechins, epicatechins,and/or procyanidins (e.g., A, B, trimer, tetramer).

Generally, one or more parts of a plant can be used to produce a plantextract including, but not limited to, the root, the stem, the leaf, theflower, the fruit, the seed, and the testa of the seed. In the presentinvention, at least the testa of the seed is used—alone or with otherplant parts—to produce the plant extract. The testa from the Anacardiumplant can be commercially obtained from various sources. The extract ofthe cashew testa can be obtained using any suitable extractiontechnique.

In this regard, one or more parts of the plant, particularly the testaof the plant, can be collected and milled. Thereafter, the milledmaterial can be extracted using a suitable solvent. The solvent can beremoved in a concentration step. For example, the extracted material canbe screened or filtered to create a supernatant and a cake. The cake canbe pressed to remove a substantial portion of the liquid, which can beadded to the supernatant. The cake can then be dehydrated and used as afiber source. The supernatant can be distilled to remove the solvent ora portion thereof, to form a plant extract liquid concentrate. Theremoved solvent can be recycled. The concentrate can be dried (e.g., byspray drying) to provide a dried plant extract. This dried plant extractcan be assayed and/or standardized as described herein. Preferably, thedried plant extract is derived from Anacardium occidentale, particularlythe testa of the plant Anacardium occidentale L.

Suitable solvents for the extraction process include water, alcohol, ormixtures thereof. Exemplary alcoholic solvents include, but are notlimited to, C₁-C₇ alcohols (e.g., methanol, ethanol, propanol,isopropanol, and butanol), hydro-alcohols or mixtures of alcohol andwater (e.g., hydroethanol), polyhydric alcohols (e.g., propylene glycoland butylene glycol), and fatty alcohols. Any of these alcoholicsolvents can be used in the form of a mixture. In one embodiment, theplant extract is extracted using ethanol, water, or a combinationthereof (e.g., a mixture of about 70% ethanol and about 30% water). Inanother embodiment, the plant extract is extracted using only water.

In one embodiment, the plant extract can be obtained using an organicsolvent extraction technique. In another embodiment, solvent sequentialfractionation can be used to obtain the plant extract. Totalhydro-ethanolic extraction techniques can also be used to obtain theplant extract. Generally, this is referred to as a lump-sum extraction.

Total ethanol extraction can also be used. This technique uses ethanolas the solvent. This extraction technique can generate a plant extracthaving fat soluble and/or lipophilic compounds in addition to watersoluble compounds.

Another example of an extraction technique that can be used to obtainthe plant extract is supercritical fluid carbon dioxide extraction(‘SFE’). In this extraction procedure, the material to be extracted maynot be exposed to any organic solvents. Rather, carbon dioxide can beused as the extraction solvent—with or without a modifier—insuper-critical conditions (>31.3° C. and >73.8 bar). Those skilled inthe art will appreciate that temperature and pressure conditions can bevaried to obtain the best yield of extract. This technique can generatean extract of fat soluble and/or lipophilic compounds, similar to atotal hexane and ethyl acetate extraction technique.

The plant extract generated in the process can include a broad varietyof phytochemicals present in the extracted material. The phytochemicalscan be fat soluble or water soluble. Following collection of the extractsolution, the solvent can be evaporated, resulting in the extract. Theplant extract can be standardized to a specified amount of a particularcompound. For example, the plant extract can be standardized to aspecified amount of an active ingredient or phytochemical. In oneembodiment, the plant extract is standardized to a catechin content ofabout 15.0 wt % or greater, based on total weight of the extract.

The amount of plant extract present in the inflammation inhibitingcomposition can depend upon several factors, including the desired levelof inflammation inhibition, the inflammation inhibiting level of aparticular plant extract or component thereof, and other factors.Preferably, the plant extract is present in an amount of from about0.005 wt % or greater, for example, from about 0.005 wt % to about 50.00wt %, based on total weight of the composition.

The anti-inflammatory composition can include one or more acceptablecarriers. The carrier can aid in enabling incorporation of the plantextract into an anti-inflammatory composition having a suitable form foradministration to a subject. A wide number of acceptable carriers areknown in the art, and the carrier can be any suitable carrier. Thecarrier is preferable suitable for administration to animals, includinghumans, and can be able to act as a carrier without substantiallyaffecting the desired activity of the plant extract and/or any activeingredient. The carrier can be chosen based upon the desiredadministration route and dosage form of the composition.

Suitable dosage forms include liquid and solid forms. In one embodiment,the composition is in the form of a gel, a syrup, a slurry, or asuspension. In another embodiment, the composition is in a liquid dosageform such as a drink shot or a liquid concentrate. In a furtherembodiment, the composition is present in a solid dosage form, such as atablet, a pill, a capsule, a dragée, or a powder. When in liquid orsolid dosage form, the composition can be in a food delivery formsuitable for incorporation into food for delivery. Examples of suitablecarriers for use in solid forms (particularly tablet and capsule forms)include, but are not limited to, organic and inorganic inert carriermaterials such as gelatin, starch, magnesium stearate, talc, gums,silicon dioxide, stearic acid, cellulose, and the like. The carrier canbe substantially inert.

As an example, silicified microcrystalline cellulose can be used as acarrier or binder. Silicified microcrystalline cellulose is a physicalmixture of microcrystalline cellulose and colloidal silicon dioxide. Onesuch suitable form of silicified microcrystalline cellulose is ProSolvSMCC® 90, available from Penwest Pharmaceutical Co., Patterson, N.J.Silicon dioxide, in addition to that provided by the silicifiedmicrocrystalline cellulose, may be added to the composition as aprocessing aid. For example, silicon dioxide can be included as aglidant to improve the flow of powder during compression in themanufacturing of solid dosage units, such as tablet.

In another embodiment, the carrier is at least a functional carrier suchas buckwheat or spelt. By the addition of functional carriers into thecomposition, additional benefits may be provided such as lower glycemicindex compared to standard carriers such as those mentioned above.Further, functional carriers can be allergen free (e.g., buckwheat), andby adding them into the production process, the botanical extracts ofthe invention may benefit from the flavonoids of these functionalcarriers, such as rutin and quercetin. Further, the high fiber contentof these functional carriers may also facilitate and regulate intestinaltransit. Finally, the added mineral benefit of selenium found in speltmay aid in metabolism.

The anti-inflammatory composition can include other inert ingredients,such as lubricants and/or glidants. Lubricants aid in the handling oftablets during manufacturing, such as during ejection from dies.Glidants improve powder flow during tablet compression. Stearic acid isan example of an acceptable lubricant/glidant.

The anti-inflammatory composition can be made in solid dosage form, suchas tablets and capsules. This form provides a product that can be easilytransported by an individual to a place of eating, such as a restaurant,and taken prior to, during, or after consumption of a foodstuff. Thecomposition can be formulated into dosage units containing suitableamounts of the plant extract and/or active ingredient that permit anindividual to determine an appropriate number of units to take basedupon appropriate parameters, such as body weight, foodstuff size, orcarbohydrate (e.g., sugar) content.

In one embodiment, the botanical extract is present in the compositionin a therapeutically effective amount, such as an amount of about 4.0μg/mL or greater, preferably from about 4.0 μg/mL to about 2000.0 μg/mL,more preferably from about 20.0 μg/mL to about 1000.0 μg/mL, even morepreferably from about 25.0 μg/mL to about 750 μg/mL. The composition canbe administered, for example, in a dosage of from about 4.0 μg/mL toabout 2000.0 μg/mL per day of the plant extract. The composition can beadministered as a single dose, or in multiple doses. In one example, thecompound is administered in up to three doses per day. For example, thecompound may be administered prior to a meal, during a meal, or after ameal. In one embodiment, the composition is a dietary supplement havinganti-inflammatory properties containing cashew testa extract in atherapeutically effective amount.

The dosage can be chosen to provide a level of inhibitory effect in asingle unit that may be effective for some individuals and/or somefoodstuffs, while also allowing for relatively simple dosage increasesto provide other levels of inhibitory effects that can be effective forother individuals and/or other foodstuffs.

The inhibiting composition can be in a form adapted for oral ingestion.This form can be configured as a single dosage form intended to providea specified dose of the plant extract. For example, the single dosageform can be a powder, a pill, a tablet, a capsule, or a drink shot. Thesingle dosage form can include, for example, from about 4.0 μg/mL toabout 2000.0 μg/mL of the plant extract.

EXAMPLES Examples—Materials and Chemical Profiling Example 1—Preparationof 70% Ethanol Extracts from Cashew Testa

Dried cashew testa powder (Anacardium occidentale L.) (60 g) was loadedinto three 100 ml stainless steel tubes and extracted twice using asolvent of 70% ethanol in DI water with a Thermo Scientific™ Dionex™ ASE350 Accelerated Solvent Extractor at a temperature of 80° C. andpressure of 1500 psi. The extract solution was filtered and collected.The combined ethanol extract solution was evaporated with a rotaryevaporator under vacuum to give a crude cashew testa extract.

The extraction results are provided in the following Table 1—

TABLE 1 Extraction of cashew testa Plant Extract Extraction PowderWeight Yield Plant Part (g) (g) (wt %) Testa 60 23.78 39.63%

Example 2—Catechin Quantification of Cashew Testa Extract

Free catechins present in the cashew testa extract were determined usinga C18 reversed-phase column (Luna® 5 μm C18(2) 100 Å LC Column 250×4.6mm, available from Phenomenex®, Torrance, Calif., US) together with anHitachi high performance liquid chromatograph with photodiode arraydetector (‘HPLC/PDA’). For mobile phase A, the solvent was 0.10%phosphoric acid (‘H₃PO₄’) in water, and for mobile phase B, the solventB was acetonitrile (‘ACN’), which was used for elution at a flow ratedof 1.0 ml/min with UV absorbance at 275 nm and a column temperature of35° C. Catechin reference standards used were from Sigma-Aldrich Co. Thereference standards were dissolved in methanol (‘MeOH’): 0.1% H₃PO₄ (1:1ratio) with catechin (C1251) at a concentration of 0.5 mg/ml andepicatechin (E1753) at 0.1 mg/ml. Testing samples were prepared at 2mg/ml in 50% MeOH in 0.1% H₃PO₄ in a volumetric flask and sonicateduntil dissolved (approximately 10 minutes), and then cooled to roomtemperature, mixed well, and filtered through a 0.45 μm nylon syringefilter. HPLC analysis was performed by injecting a 20 μl sample into theHPLC. Table 2 below provides the gradient table of HPLC analyticalmethod—

TABLE 2 Gradient Table of HPLC Analytical Method Mobile Mobile Time(min) Phase A Phase B 0.0 85.0 15.0 7.0 85.0 15.0 12.0  10.0 90.0 16.5 10.0 90.0 16.6  85.0 15.0 24.0  85.0 15.0

HPLC Catechin quantification results in cashew testa extract provided acatechin content of 9.40% and an epicatechin content of 6.12%, for atotal catechin content of 15.52% by weight, based on total weight of theextract. Accordingly, the cashew testa extract can be standardized to atotal catechin content of about 15.00% or greater by weight, based ontotal weight of the extract. The HPLC chromatogram for cashew testaextract at 275 nm wavelength is provided in FIG. 3.

Example 3—Chemistry Profiling of Cashew Testa Extract

Flavonoid compounds present in the cashew testa extract were determinedusing ultra high pressure liquid chromatography (‘HPLC’) and massspectrometry (ACQUITY® UPLC I-Class and XEVO® GS-XT-QT of system, bothavailable from Water Corporation, Milford, Mass. USA). Column used wasan ACQUITY® UPLC HSS T3 2.1×100 mm, 1.8 μm, with a column temperature of40° C. and a sample temperature of 15° C. For the mobile phase, SolventA was 10% acetonitrile (‘ACN’) in water (0.1% Formic Acid), and SolventB was ACN. The acquisition range was 100-1500 Daltons (‘Da’), and theacquisition mode was electrospray ionization (‘ESI’) (−). Table 3 belowprovides the HPLC conditions—

TABLE 3 HPLC conditions for analyzing cashew testa extract Run TimeInjection (min) Volume (μL) Concentration 20.00 2.00 1 mg/mL

Peak identification was based on accurate mass only. Digalloyl catechin,catechin and epicatechin were identified as the major components forcashew testa extract. Procyanidins were detected in the extract as well,including A- and B-type procyanidins, procyanidin tetramer, andprocyanidim trimer, with B-type procyanidins being the major componentof the procyanidins. Compounds identified, in addition to those justmentioned, included digalloyl catechin, vaccihein A,6″-p-coumaroylprunin, and dunalianoside B, among others. LC/MS andLC/PDA chromatograms of cashew testa extract obtained from the analysisare illustrated in FIG. 4.

Examples—Bioassay

Extracts of cashew testa were prepared with food-grade ethanol, and thenfiltered and dried as described above. Research grade reagents were usedfor the rest of the assay preparations. Extracts were dissolved indimethyl sulfoxide (‘DMSO’) to a final concentration of 50 mg/mL, andthen diluted in appropriate buffer for each bioassay to workingconcentrations.

Example 4—COX-1 and COX-2 Inhibition

Cashew testa extract was tested for COX-1 inhibition using thecyclooxygenase-1 (‘COX-1’) Inhibitor Screening Kit (catalog #K548) fromBioVision (Milpitas, Calif., US). This screening kit measures theproduction of the organic peroxide prostaglandin G2, a product generatedby the COX enzyme, over a time course. Extracts were dissolved toworking concentrations in DMSO with COX Assay Buffer to a finalconcentration of 5% DMSO. SC-560 COX-1 inhibitor was used as a positivecontrol. COX-1 enzyme was reconstituted in sterile water and stored at-80° C. COX cofactor and arachidonic acid solutions were diluted justprior to use. COX probe, COX cofactor, and COX-1 enzyme solution wereadded to the test samples and controls before the arachidonic acidsolution was quickly added to start the reaction. Fluorescence wasmeasured every minute for 10 minutes at the following wavelengths:excitation −535 nm, emission 590 nm. The slope of the linear portion ofthe curve (FIG. 5) was deduced and percent inhibition of the uninhibitedcontrol was calculated. Referring to FIG. 5, various degrees of COX-1inhibition were observed, depending on the concentration of cashew testaextract. Cashew testa extract COX-1 inhibition was observed to be fromabout 4 μg/mL to at least about 2000 μg/mL, more particularly from about15 μg/mL to about 250 μg/mL, with an IC₅₀ of 32 μg/mL.

Cashew testa extract was tested for COX-2 inhibition using thecyclooxygenase-2 (‘COX-2’) Inhibitor Screening Kit (catalog #K547) fromBioVision (Milpitas, Calif., US). This screening kit measures theproduction of the organic peroxide prostaglandin G2, a product generatedby the COX enzyme, over a time course. Extracts were dissolved toworking concentrations in DMSO with COX Assay Buffer to a finalconcentration of 10% DMSO. Celecoxib nonsteroidal anti-inflammatory drug(‘NSAID’) was used as a positive control. COX-2 enzyme was reconstitutedin sterile water and stored at −80° C. COX cofactor and arachidonic acidsolutions were diluted just prior to use. COX probe, COX cofactor, andCOX-1 enzyme solution were added to the test samples and controls beforethe arachidonic acid solution was quickly added to start the reaction.Fluorescence was measured every minute for 10 minutes at the followingwavelengths: excitation −535 nm, emission 590 nm. The slope of thelinear portion of the curve (FIG. 6) was deduced and percent inhibitionof the uninhibited control was calculated. Referring to FIG. 6, variousdegrees of COX-2 inhibition were observed, depending on theconcentration of cashew testa extract. Cashew testa extract COX-2inhibition was observed to be from about 4 μg/mL to at least about 2000μg/mL, more particularly from about 30 μg/mL to about 250 μg/mL, with anIC₅₀ of 86 μg/mL. Accordingly, based on the results presented herein,cashew testa extract may have reasonable activities in ameliorating theactivity or release of COX-1 and COX-2, suggesting its usage ininflammatory diseases mediated by COX-1 and COX-2.

Example 5—5-LOX Inhibition

Cashew testa extract was tested for 5-LOX inhibition using theLipoxygenase Inhibitor Screening Assay Kit (available from CaymanChemical, Ann Arbor, Mich., US) and potato 5-Lipoxygenase enzyme(available from Cayman Chemical). This kit measures hydroperoxidesproduced in the lipoxygenation reaction.

The extracts were dissolved in methanol to final working concentrations.5-LOX enzyme, Chromagen, and Linoleic Acid solutions were preparedimmediately before use. Nordihydroguaiaretic acid (‘NDGA’) was used as apositive control. 5-LOX enzyme was added to the test samples andcontrols and incubated for five minutes at room temperature to allow forenzyme/inhibitor interaction. Linoleic acid substrate was added to theplate to initiate the reaction, and the plate was then shaken at roomtemperature for 10 minutes. Chromagen was added to visualize thehydroperoxides formed during the reaction and the plate was shaken atroom temperature for another five minutes. The absorbance was then readat 492 nm. Percent inhibition of the extract concentration wascalculated in comparison to the uninhibited control wells.

Cashew testa extract was tested for its 5-LOX inhibition activity at 10different concentrations (0.7, 1.5, 3.0, 6.0, 11.9, 15.6, 31.2, 62.5,125.0 and 250.0 μg/mL). NDGA was used as a positive control at 100 μMwith a 100% 5-LOX enzyme inhibition. Referring to FIG. 7, cashew testaextract 5-LOX inhibition was observed to be from about 32 μg/mL to atleast about 250 μg/mL, more particularly from about 32 μg/mL to about125 μg/mL, with an IC₅₀ of 55 μg/mL observed for the cashew testaextract. Accordingly, based on the results presented herein, cashewtesta extract may have reasonable activities in ameliorating theactivity or release of 5-LOX, suggesting its usage in inflammatorydiseases mediated by 5-LOX.

Example 6—HMGB1 Inhibition

HMGB1 Experimental Procedure—

Cell Culture. Murine macrophage-like cells (available as RAW 264.7(ATCC® TIB-71™) from American Type Culture Collection (ATCC), Manassas,Va., US) were cultured in Dulbecco's Modified Eagle's Medium (‘DMEM’)((DMEM) (ATCC® 30-2002™), from American Type Culture Collection (ATCC),Manassas, Va., US) supplemented with 10% fetal bovine serum (fromAtlanta Biologicals, Lawrenceville, Ga., US). The cells were maintainedunder normoxic conditions (5% CO₂/21% O₂), allowed to grow to 70-80%confluency, and subcultured every two (2) days.

Extract/Drug Preparation. Cashew testa extract was stored in powder format −20° C. Prior to treating cells with extract, a stock solution volumeof the extract was adjusted to a final concentration of 50 mg/mL indimethyl sulfoxide (‘DMSO’) (from AMRESCO, Inc., Solon, Ohio, US) andstored at −20° C. Extract was diluted to a final concentration of 0.25mg/mL in serum-free Opti-MEM™ I medium (from Gibco-BRL, Gaithersburg,Md., US) and filtered sterilized by 0.2 μm PES syringe filter (from VWR,Radnor, Pa., US). Sodium salicylate (from AMRESCO, Inc., Solon, Ohio,US) was prepared at 2-20 μM as a positive control, which can attenuatehyperoxia-induced HMGB1 release from macrophages.

Hyperoxia Exposure. The exposure of murine macrophage RAW 264.7 cells tohyperoxia was achieved in sealed, humidified Plexiglas chambers (fromBillups-Rothenberg, Del Mar, Calif., US) flushed with 95% O₂/5% CO₂ at37° C. for 24 hours.

HMGB1 ELISA. To determine the levels of extracellular HMGB1, RAW 264.7cells were cultured in serum-free Opti-MEM™ I medium (from Gibco-BRL,Gaithersburg, Md., US) in 6-well plates and were kept at either 21% O₂(room air) or exposed to 95% O₂ with or without the cashew testa extractfor 24 hours. After hyperoxic exposure, the levels of HMGB1 in theculture media were measured by ELISA (enzyme-linked immunosorbentassay). Cell culture media was collected and pelleted at 500 g for 5minutes at 4° C. Equal volumes of cell culture supernatant were thenapproximately 6-x's concentrated using Amicon Ultra-4 centrifugal units(from EMD Millipore, Burlington, Mass., US). Just after concentration,equal volumes of cell culture supernatant concentrate were loaded onto a96-well plate for determination of HMGB1 by ELISA according tomanufacturer's instructions (from Chondrex, Inc., Redmond, Wash., US).Plate absorbances were determined by reading the optical density (‘OD’)value at 450 nm (with 630 nm used as a reference) on a Thermo MultiscanEx microplate reader (from Thermo Scientific, Waltham, Mass., US). HMGB1levels were determined in sample cell culture supernatant by comparisonto a standard curve and further corrected by applying concentrationfactors.

Statistical Analysis. Data was presented as the mean ±standard error ofthe mean (SEM) of one to three independent experiments. Data wasanalyzed by use of one-way analysis of variance (ANOVA) using Fisher'sLeast Significant Difference (‘LSD’) post-hoc analysis and GraphPadPrism version 6 software (from GraftPad Software, La Jolla, Calif., US).A P-Value of <0.05 was considered statistically significant.

HMGB1 Experimental Results—

Referring to FIG. 8, it is seen that hyperoxia (‘O2’) resulted in asignificant increase in HMGB1 level compared to cells treated with 21%O₂ (‘RA’). These elevated levels of HMGB1 were reduced closer to normallevels (cells exposed to room air (RA) with no treatment) as a result oftreatment with cashew testa extract (‘CT’). Similar reductions wereobserved for the positive control sodium salicylate (‘SS’). Reductionsfor both treatment groups (SS and CT) were statistically significant.Accordingly, based on the results presented herein, cashew testa extractmay have reasonable activities in ameliorating the activity or releaseof HMGB1, suggesting its usage in inflammatory diseases mediated byHMGB1.

The above data illustrates that the botanical extract of the testa ofAnacardium occidentale L. has one or more compounds that exhibitanti-inflammatory activity. More particularly, the cashew testa extractmay have reasonable activities in ameliorating the activity or releaseof COX-1, COX-2, 5-LOX, and/or HMGB1.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Further, unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs. Consequently, it is not intended that this inventionbe limited to the specific embodiments disclosed herein, but that itcover all modifications and alternatives coming within the true scopeand spirit of the invention as embodied in the attached claims.

We claim:
 1. A method of inhibiting inflammation in a subjectcomprising: administering to the subject a composition comprising thebotanical extract of the testa of Anacardium occidentale L., wherein thebotanical extract has been standardized to a catechin content of about10.0 w/w % or greater, based on total weight of the extract, and whereinthe botanical extract is present in the composition in an amount of fromabout 25.0 μg/mL to about 250.0 μg/mL.
 2. The method of claim 1, whereinthe composition inhibits COX-1 activity.
 3. The method of claim 1,wherein the composition inhibits COX-2 activity.
 4. The method of claim1, wherein the composition inhibits 5-LOX activity.
 5. The method ofclaim 1, wherein the composition inhibits HMGB 1 activity.
 6. The methodof claim 1, wherein the composition is a dietary supplement.
 7. Themethod of claim 6, wherein the dietary supplement is in solid dosageform.
 8. The method of claim 1, wherein the composition is a topicalcomposition.